Enhanced AP-1 and NF-kappaB activities and stability of interleukin 8 (IL-8) transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells.
Inhibition of protein synthesis may result in superinduction of short-lived transcripts and has been attributed variably to stabilization of transcripts and/or increased gene transcription. Little is known about the kinetics of these processes and relevant transcriptional elements have not been identified. In this study, we describe superinduction of interleukin 8 (IL-8) mRNA, an important inflammatory mediator, in lung epithelial-like H292 cells and identify the underlying molecular mechanisms and their kinetics. Cycloheximide (CHI, 10 microg/ml), an inhibitor of protein synthesis, maximally increased IL-8 mRNA levels 30-fold in H292 cells. Tumour necrosis factor alpha (TNF-alpha), which induced IL-8 mRNA 3-fold, synergized with CHI causing a 150-fold increase at 6 h. CHI early on increased the stability of IL-8 mRNA (from 40 min in cells cultured with medium to more than 4 h with CHI). CHI also increased transcription as shown by transfection with IL-8 promoter constructs. Truncated and mutated constructs identified NF-kappaB and AP-1 binding sites as primary cis-acting elements in IL-8 gene transcription and IL-8 mRNA superinduction. Electrophoretic mobility shift assays indicated that CHI increased NF-kappaB and prolonged AP-1 DNA-binding activities and that the synergism of TNF-alpha and CHI on IL-8 mRNA expression was paralleled by a further increase of AP-1 DNA-binding activity. This synergism was still noticed when 4 h elapsed between the addition of CHI and that of TNF-alpha. Taken together, our results indicate that CHI interferes with both post-transcriptional and transcriptional repressive mechanisms of IL-8 mRNA expression.
Roger
T
T
Department of Pulmonology, Academic Medical Centre, Meibergdreef 9, P.O. Box 22700, 110 DE Amsterdam, The Netherlands.
Out
T
T
Mukaida
N
N
Matsushima
K
K
Jansen
H
H
Lutter
R
R
D002467:Cell Nucleus / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D016209:Interleukin-8 / Q000502:physiology*; D008168:Lung / Q000502:physiology*; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D004268:DNA-Binding Proteins; D016209:Interleukin-8; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide